CA1122119A - Process for the determination of the thyroxine- binding index in serum - Google Patents
Process for the determination of the thyroxine- binding index in serumInfo
- Publication number
- CA1122119A CA1122119A CA000326897A CA326897A CA1122119A CA 1122119 A CA1122119 A CA 1122119A CA 000326897 A CA000326897 A CA 000326897A CA 326897 A CA326897 A CA 326897A CA 1122119 A CA1122119 A CA 1122119A
- Authority
- CA
- Canada
- Prior art keywords
- thyroxine
- serum
- enzyme
- container
- solid phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/81—Tube, bottle, or dipstick
Abstract
ABSTRACT OF THE DISCLOSURE
The thyroxine-binding index in serum is determined by mixing a serum sample under investigation, with a definite amount of thyroxine and of a determinable enzyme, for example peroxidase; the resulting solution is contacted with anti-thyroxine antibodies in a solid phase, a liquid phase is separated from the solid phase and the activity of the determinable enzyme used is measured in one of the phases.
The thyroxine-binding index in serum is determined by mixing a serum sample under investigation, with a definite amount of thyroxine and of a determinable enzyme, for example peroxidase; the resulting solution is contacted with anti-thyroxine antibodies in a solid phase, a liquid phase is separated from the solid phase and the activity of the determinable enzyme used is measured in one of the phases.
Description
~22~
The present invention is concerned with a process and a reagent Eor the determination of the thyroxine-binding index in serum~
Because of the wide extent of thyroid gland investigations, thyroid gland diagnosis is of especial importance in clinical chemistry. The hasic ln vitro thyroid gland diagnosis includes the determination of the total thyroxine present in the serum (total T4~, as well as the determination of the concentration of the thyroxine-binding globulin (TBG), which takes place with the help of the so-called triiodothyronine uptake (T3 uptake) or thyroxine-binding index (TBI test).
The above-mentioned T3 uptake test is used to - determine the residual binding capacity ~BK) of carrier proteins for thyroid gland hormones in the serum~
These proteins are thyroxine~binding globulin (TBG), to which is bound about 60% of the total T4 amount present, as well as thyroxine~binding prealbumin (TBPA) and albumin. Consequently, this residual binding capacity is substantially a ~ues-tion of the binding of the .
thyroid gland hormones on to the TBG. Thus, in the case of hyperthyreosis with, for example, an increased T4 content, more binding sites of the carrier protein are occupied by the thyroid gland hormones T~ and T3, whereas, in ~he case of h~pothyreosis, less binding sites of the carrier protein are occupied by the thyroid gland hormones T4 and T30 : ' ~
. ., In the case of a known process for the d~termin-ation of the TBI, a definite amount of radioactive T3 is mixed with a ser~ sample and an anion exchanger.
The free binding sites of the carrier protein and of the anion exchanger compete for the radioactive T3. The more unoccupied binding sites are present on the carrier protein, then the more radioactive T3 is bound thereon, the remainder going on to the ion exchanger. Evaluation is carried out by measuring the radioactivity either on the ion exchanger or in the solution. The ratio of the radioactivity in the solution, which corresponds to that amount of radioactive T3 which is bound on to the carrier proteins of the serum to be investigated, to the radio-activity in the solution of a st:andard sample, multiplied with a specific factor for the standard gives the so-called thyroxine binding index TBI.
A disadvantage of this known process is the nec-essity of having to use radioactive substances. This require~, on the one hand, eXperlSiVe and complicated measurement devices and, on the other hand, the handling of radioactive reagents, which is made difficult due to the legal requirements because of the potential damage to health of these substances.
Therefore, it is an object of the present invention to provide a process for the TBI determination which does not suffer from these disadvantages and enables, with the use of ~imple measuring devices present in every laboratory, , -~2~
a dependable determination of the TBI index for thyroid gland diaynosis to be carried out.
Thus, according to the present invention, there is provided a process for the determination of the ~hyroxine-binding index in serum, wherein a serum sample to be investigated i8 mixed with a definite amount of thyroxine and a definite amount of a determinable enzyme covalently ~ound to -thyroxine~ the solution obtained is contacted with antithyroxine antibodies present in the solid phase, the liquid phase is sepaxated from the solid phase and the activity of the determinable enæyme employed is measured on one of the phasesO
The present invention is based upon the consider-ation that replacement of the radioactive marking in the known T3 uptake test by an enz~me markin~, with the use of an easily determinable enzyme as marker, could solve the problem. However, experiments which have been carried out showed that it is not possible, in the case of the known ~3 uptake test, to replace radioactively 3_~ 3 I125) by enzyme-marked T3 or T4, which would be pr~ferable. Th~s, it transpired that enz~me-marked T3 or T4 is not bound by the sites on the serum proteins capable of binding. It is assumed tha~ this is due to the voluminous enzyme residue which sterically hinders bindiny.
Su~prisingly, however, we have found that enzyme-marked T4 is able to compete with non-enzyme-marked T4
The present invention is concerned with a process and a reagent Eor the determination of the thyroxine-binding index in serum~
Because of the wide extent of thyroid gland investigations, thyroid gland diagnosis is of especial importance in clinical chemistry. The hasic ln vitro thyroid gland diagnosis includes the determination of the total thyroxine present in the serum (total T4~, as well as the determination of the concentration of the thyroxine-binding globulin (TBG), which takes place with the help of the so-called triiodothyronine uptake (T3 uptake) or thyroxine-binding index (TBI test).
The above-mentioned T3 uptake test is used to - determine the residual binding capacity ~BK) of carrier proteins for thyroid gland hormones in the serum~
These proteins are thyroxine~binding globulin (TBG), to which is bound about 60% of the total T4 amount present, as well as thyroxine~binding prealbumin (TBPA) and albumin. Consequently, this residual binding capacity is substantially a ~ues-tion of the binding of the .
thyroid gland hormones on to the TBG. Thus, in the case of hyperthyreosis with, for example, an increased T4 content, more binding sites of the carrier protein are occupied by the thyroid gland hormones T~ and T3, whereas, in ~he case of h~pothyreosis, less binding sites of the carrier protein are occupied by the thyroid gland hormones T4 and T30 : ' ~
. ., In the case of a known process for the d~termin-ation of the TBI, a definite amount of radioactive T3 is mixed with a ser~ sample and an anion exchanger.
The free binding sites of the carrier protein and of the anion exchanger compete for the radioactive T3. The more unoccupied binding sites are present on the carrier protein, then the more radioactive T3 is bound thereon, the remainder going on to the ion exchanger. Evaluation is carried out by measuring the radioactivity either on the ion exchanger or in the solution. The ratio of the radioactivity in the solution, which corresponds to that amount of radioactive T3 which is bound on to the carrier proteins of the serum to be investigated, to the radio-activity in the solution of a st:andard sample, multiplied with a specific factor for the standard gives the so-called thyroxine binding index TBI.
A disadvantage of this known process is the nec-essity of having to use radioactive substances. This require~, on the one hand, eXperlSiVe and complicated measurement devices and, on the other hand, the handling of radioactive reagents, which is made difficult due to the legal requirements because of the potential damage to health of these substances.
Therefore, it is an object of the present invention to provide a process for the TBI determination which does not suffer from these disadvantages and enables, with the use of ~imple measuring devices present in every laboratory, , -~2~
a dependable determination of the TBI index for thyroid gland diaynosis to be carried out.
Thus, according to the present invention, there is provided a process for the determination of the ~hyroxine-binding index in serum, wherein a serum sample to be investigated i8 mixed with a definite amount of thyroxine and a definite amount of a determinable enzyme covalently ~ound to -thyroxine~ the solution obtained is contacted with antithyroxine antibodies present in the solid phase, the liquid phase is sepaxated from the solid phase and the activity of the determinable enæyme employed is measured on one of the phasesO
The present invention is based upon the consider-ation that replacement of the radioactive marking in the known T3 uptake test by an enz~me markin~, with the use of an easily determinable enzyme as marker, could solve the problem. However, experiments which have been carried out showed that it is not possible, in the case of the known ~3 uptake test, to replace radioactively 3_~ 3 I125) by enzyme-marked T3 or T4, which would be pr~ferable. Th~s, it transpired that enz~me-marked T3 or T4 is not bound by the sites on the serum proteins capable of binding. It is assumed tha~ this is due to the voluminous enzyme residue which sterically hinders bindiny.
Su~prisingly, however, we have found that enzyme-marked T4 is able to compete with non-enzyme-marked T4
2~
!
for anti-T4 antibodies which are present in the solid phase. Therefore, in the process according to the present invention, on the one hand, the bindable places of the sexum protein and~ose of the insoluble anti-T4 antibodies compete for the T4, whereby the more T4 is bound on to the anti-T4 antibodies, the less free bind-ing sites are present in the serum. At the same timeO
enzyme-marked T4 competes wlth non marked T4 for the anti-T4 antibodies. Therefore, the more enzyme-marked T4 is bound on to the solid phase, the greater is the thyroxine~binding capacity of the serum and thus the thyroxine--binding index TBI. ~herefore, the measured enzyme activity in the ~olid phase represent~ a direct measure for the magnitude of the TBI. However, the activity can also be detenmined in the liquid phaseq - According to the present inventlon, use can be made of any determinable enzyme which, without loss of its enzymatic activity; can be bound to thyroxine, those mar~ing enz~nes being preferably used, the ac~-ivity of which can easily be determined by an optical :
test and especially by a colour r~action in visible - light or by change of the NADH concentration. Typical - example~ of marking enzymes which can be used include`
peroxidase, glucose oxidase, a- and ~-glactosidase, glucoamylase, invertase and alkaline phosphatase~
For all the abo~e-mentioned enzymes, simple and rapid detèrmination methods are known, with regard to , .
which refereIIce is made, for exam~le, to H.U.Bergmeyer's "Methoden der enzymatischen Analyse", pub. Verlag Chemie, 3rd edition, 1974. The anti-T4 antibody present in the solid phase can be free of a carrier, for example rendered insoluble by cross-linking with a polyfunctional reagent, such as a dialdehyde, dioxide or the likeO How-ever, the antibody is preferably bound to a solid carrier material. The solid carrier for the anti-T4 antibody can be any material which is inert towards the substances employed in the test on to which ~he mentioned antibody can be bound. We have found that, in the case of numerous materials, the anti-T~ antibody can be sufficiently securely bound by adsorptive forces to the surface of the carrier material. ~owever, if desired, the antibody can also be fixed by methods conventionally used for fixing biologically-active proteins on to solid carrier materials, for example vla covalent bonds.
Typical examples of this include the activation of the carrier surface by the cyanogen bromide method, by protein copolymerisation and by surface cross-linking of the antibody on to the carrier by means of poly-functional cross~linking agents, such as glutardialdehyde or the like, these methods being well known.
According to the present invention, it is-prefer-able to use containers, such as test tubes, the inner wall of which is coated with the antibody. Typical examples of container materials which can be used include polystyrene and styrene-acrylonitrile copolymers, as well as other synthetic resins and glass. In the case of synthetic resins based on styrene, a sufficient coating can be obtained merely by leaving an antibody solution in the container for some time.
The above remarks apply analogously to carrier materials which are not in the form of containers but are, for example, in the form of particles which can be used for column packings~
~ nti-T4 antobodies are prepared by the u~ual methods for obtaining antibodies against hormones, the use of a conjugate of T4 on to an appropriate carrier protein being preferred as immunogen~ Of the large number of well-known carrier proteins, bovine serum albumin (BSA) is especially preferred. ~he carrier proteins are coupled chemically with the T4, ~or example with glutardialdehyde in a weakly alkaline medium, followed by reduction with sodium borohydride.
Another coupling method which can be used is the reaction with morpholi~o-dicyclohexyl-carbodiimide.
Experimental animals for the administration of the so obtained T4 i~munogen include, for example, rabbits or sheep but other animal~ can also be used.
The preparation of T4 marked with peroxidase, wh~ch is preferred according to the present i~vention, can be carried out by the reaction of tert.-butyloxy-carbonyl~thyroxine-N-hydroxysuccinimide with peroxidase ~22~
(POD~, followed by chromatographic purification of the product obtained. Most other enzymes coming into con-sideration can also be bound in the same manner, as well as with the use of other known protein-chemical processes.
As mentioned above, according to the present invention, a definite amount of T4 is used. We have found that this amount is preferably from 100 to 500 ng.T4/ml. of serum, although greater or smaller amounts can also be used. However, the above-mentioned range of amounts covers practically all serum compositions which occur and, consequently, suf~ices for hyperthyroid as well as for hypothyroid sera. The ~mount of enzyme-marked T4 added depends upon the nature of the particular enzyme employed and especially upon the specific activity - of this enzyme which can still be easily determined. In the case of the preferred marking with POD, such an amount is preferably added that the actual test mixture contains from about ] to about 200 mU POD/mlO
The reaction mixture of serum, buffer, T4 and en~yme-mar~ed T4 is contacted with insoluble and prefer-ably carrier-bound anti-T~ antibody for a time which is sufficiently long to give a constant binding rate of the marked T4 on to the antibody. The period of time depends, to a certain extent, upon the conditions with regard to pH value, temperature and concentration. In general, a period from about 30 minutes to about 2 hours is sufficient. ~en incubation is completed, the sol-ution is separated from the carrier, for example by `
- 9 - ~
pouring out of the container used, the carrier ls washed with water and subsequently the determination of the carrier-bound enzyme activity is carried out. In the case of the preferred embodiment using a container carrier and using POD as the marking enzyme, there is added, for example, hydrogen peroxide and ABTS [2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate)] in a buffer solution, khe extinction difference being deter-mined at a measurement wavelength of 405 nm.
The presenk invention also provide~ a test kit for carrying out the process according to the present invention, which test kit consists essentially of thyroxine, enzyme-marked thyroxine, anti-thyroxine anti-body in the form of insoluble solid material, a bu~fer and a rea~ent for the determination of the enzyme act-ivity. Buffers which can be used are those with which a pH value of from 7.5 to about 9~3 can be adjusted, a pH value of from 8,0 to 9.O being preferredO Typical examples of buffers which can ~e used in the g~ven range include phosph~te buffer, tris buffer, borax buffer and barbital-huffer, 0.05 to 0.5M b~rbital buffer being preferred.
The test kit according to the present invention ~an also contain conventional stabilising agents, such as bovine serum albumin, carbohydrates and/or glycerol, as well as immune reaction-promoking additives.
- As enzyme~marked T4, the test kit according to . J
. .
2~
the present invention preferably contains thyroxine-pero~idase. The reagent for the determination of the enzyme activity can then, in this preferred case, com-prise hydrogen peroxide or a compound which provides hydrogen peroxide, such as sodium perborate or per-hydrite, as well as 2~2'-azino-di-(3-ethylbenzthiazoline-6~sulphonate~, usually referred to as ABTS, and also an appropriate buffer. A typical example of an appropriate buffer is phosphate-citrate buffer (pH 4.5 to 6.0).
Another reagent which can be used for this embodiment comprises phosphate buffer tpH 7.0), guaiacol and hydrogen peroxide.
A special embodiment of the preferred test kit o~
the present invention comprises or contains:
thyroxine 100 - 440 ng./ml.
thyroxine-POD 5 - 100 mU/ml.
barbital buffer (pH 8.6)Ool - 0.2M
bovine serum albumin 0.2%
anti-thyroxine antibody (calculated without carrier) 1 - 0.05 g./ml.
hydrogen peroxide 0.5 - 5 mM/l.
ABTS 5 - 50 mM/l.
phosphate-citrate buffer 0.1 0.2M
For the preparation of the enzyme-marked thyroxine, it is preferable to ~tart from tert.-butylo~ycarbonyl-thyroxine~N-hydroxysuccinimide, the reaction taking place in buffer/dimethylformamide solution (1:1 v/v)~
--ll The buffer used is that which is best suited for the particular enæyme used. Phenylbutylamine-sepharose has proved to be useful for the purification of the conjugate.
Thus, the present invention provides a simple process, which can be carried out with conventional laboratory devices, for the determination of the thyroxine-binding index of serum, the exactitude of which corres-ponds to that of the known methods using radioactive marking but does not suffer from their disadvantages.
The following Example is given for the purpose of illustrating the present invention:-Example.
A. Preparation of anti-thyroxine antibodies.
Thyroxine is coupled to ~ovine serum albumin in aqueous ~olution (pH 10) by the addition of 1.9% by wei~ht glutardialdehyde. The Schiff base bond is then reduced with excess sodium borohydride and the T4 immunogen obtained is purified by chromatography. The product obtained is dialysed and then administered to the experimental animals~
~ .
POD i~ reacted with a 10 fold molar excess of tert.-butyloxycarbonylthyroxine-N-hydroxysuccinimide in dimethylformamide/phosphate buffer lpH 8.5) tl-l v/v).
The dimethylformamide is then dialysed off and the aqueous solution i3 passed over phenylbutyl~mine-sepharose~ The column is washed with tris-sodium chloride buffer and then with 1~ sodium chloride-containiny ethylene glycol/water mi~ture ~1:1 v/v).
The eluate is stirred for 2 hours with 0.5M hydroxylamine and subsequently dialysed against phosphate buffer. The soluti~n obtained i5 mixed with bovine serum albumin and lyophilisedO
C. _reparation of carrier-bound anti-T antibodies.
Anti-T4 antiserum is obtained in known manner from the immunised experimental animals and precipitated with ammonium sulphate. The precipitate is taXen up with 0.04M phosphate buffer 1:6000. 1.5 ml. of the solution so obtained is left to stand overnight in test tubes made of styrerle-acrylonitrile polymer (Luran~, then sucked out and washed with physiological sodium chloride solutio~ which contains 1% BSA and thereafter dried.
nation.
Into the anti-T4 antibody-coated test tubes obtained according to C above are pipetted 10 ~1. serum and sub~equently 1 ml. of a reagent which contains 280 ng T4/ml., 10 mU~ml~ T4-POD in 0.12M barbital buf~er ~pH 8.6) and 0. 2Yo bo~ine serum albumin. After standing ~or 2 hours at ambient ternperature, the test tubes are emptied by sucking out and the POD reagent is placed therein, the latter comprising 1.47 r~M/l.
hydrogen peroxide and 14 mM/l. ABTS in 0~2M phosphate-cit,rate huf~er (pH 5.0). The colour change which occurs is measured at 405 nm.
~ J ~ ~a rl~
For the evaluation, a calibration line is produced with 2 standards of differing T~-~inding capaity which are treated in the same way as the samples, the reciprocal values of the extinctions measured for these standards being plotted either against the T4 amounts bound by the standards or directly against the previously deter-mined TBI values of these standards.
E. Use of the method for the investiqation of human sera.
83 Human sera samples are comparatively investigated y the process according to the~present invention and with method using the use of a commercially available/T3-I125. If the investigated sera are classified on the basis of the particular TBI values obtained with re~erence to the normal range valid for the method in question in those sera with reduced, normal and lncreased T~-binding capacity, then a satisfactory agreement results between both methods (see Table 1).
Comparison of khe process according to the present invention for the determination o~ the TBI (A) with a conventional TBI radio-assay ~B), using human serum samples (n = 83) ~ .
1) reduced 2) normal ) T4-binding capacity
!
for anti-T4 antibodies which are present in the solid phase. Therefore, in the process according to the present invention, on the one hand, the bindable places of the sexum protein and~ose of the insoluble anti-T4 antibodies compete for the T4, whereby the more T4 is bound on to the anti-T4 antibodies, the less free bind-ing sites are present in the serum. At the same timeO
enzyme-marked T4 competes wlth non marked T4 for the anti-T4 antibodies. Therefore, the more enzyme-marked T4 is bound on to the solid phase, the greater is the thyroxine~binding capacity of the serum and thus the thyroxine--binding index TBI. ~herefore, the measured enzyme activity in the ~olid phase represent~ a direct measure for the magnitude of the TBI. However, the activity can also be detenmined in the liquid phaseq - According to the present inventlon, use can be made of any determinable enzyme which, without loss of its enzymatic activity; can be bound to thyroxine, those mar~ing enz~nes being preferably used, the ac~-ivity of which can easily be determined by an optical :
test and especially by a colour r~action in visible - light or by change of the NADH concentration. Typical - example~ of marking enzymes which can be used include`
peroxidase, glucose oxidase, a- and ~-glactosidase, glucoamylase, invertase and alkaline phosphatase~
For all the abo~e-mentioned enzymes, simple and rapid detèrmination methods are known, with regard to , .
which refereIIce is made, for exam~le, to H.U.Bergmeyer's "Methoden der enzymatischen Analyse", pub. Verlag Chemie, 3rd edition, 1974. The anti-T4 antibody present in the solid phase can be free of a carrier, for example rendered insoluble by cross-linking with a polyfunctional reagent, such as a dialdehyde, dioxide or the likeO How-ever, the antibody is preferably bound to a solid carrier material. The solid carrier for the anti-T4 antibody can be any material which is inert towards the substances employed in the test on to which ~he mentioned antibody can be bound. We have found that, in the case of numerous materials, the anti-T~ antibody can be sufficiently securely bound by adsorptive forces to the surface of the carrier material. ~owever, if desired, the antibody can also be fixed by methods conventionally used for fixing biologically-active proteins on to solid carrier materials, for example vla covalent bonds.
Typical examples of this include the activation of the carrier surface by the cyanogen bromide method, by protein copolymerisation and by surface cross-linking of the antibody on to the carrier by means of poly-functional cross~linking agents, such as glutardialdehyde or the like, these methods being well known.
According to the present invention, it is-prefer-able to use containers, such as test tubes, the inner wall of which is coated with the antibody. Typical examples of container materials which can be used include polystyrene and styrene-acrylonitrile copolymers, as well as other synthetic resins and glass. In the case of synthetic resins based on styrene, a sufficient coating can be obtained merely by leaving an antibody solution in the container for some time.
The above remarks apply analogously to carrier materials which are not in the form of containers but are, for example, in the form of particles which can be used for column packings~
~ nti-T4 antobodies are prepared by the u~ual methods for obtaining antibodies against hormones, the use of a conjugate of T4 on to an appropriate carrier protein being preferred as immunogen~ Of the large number of well-known carrier proteins, bovine serum albumin (BSA) is especially preferred. ~he carrier proteins are coupled chemically with the T4, ~or example with glutardialdehyde in a weakly alkaline medium, followed by reduction with sodium borohydride.
Another coupling method which can be used is the reaction with morpholi~o-dicyclohexyl-carbodiimide.
Experimental animals for the administration of the so obtained T4 i~munogen include, for example, rabbits or sheep but other animal~ can also be used.
The preparation of T4 marked with peroxidase, wh~ch is preferred according to the present i~vention, can be carried out by the reaction of tert.-butyloxy-carbonyl~thyroxine-N-hydroxysuccinimide with peroxidase ~22~
(POD~, followed by chromatographic purification of the product obtained. Most other enzymes coming into con-sideration can also be bound in the same manner, as well as with the use of other known protein-chemical processes.
As mentioned above, according to the present invention, a definite amount of T4 is used. We have found that this amount is preferably from 100 to 500 ng.T4/ml. of serum, although greater or smaller amounts can also be used. However, the above-mentioned range of amounts covers practically all serum compositions which occur and, consequently, suf~ices for hyperthyroid as well as for hypothyroid sera. The ~mount of enzyme-marked T4 added depends upon the nature of the particular enzyme employed and especially upon the specific activity - of this enzyme which can still be easily determined. In the case of the preferred marking with POD, such an amount is preferably added that the actual test mixture contains from about ] to about 200 mU POD/mlO
The reaction mixture of serum, buffer, T4 and en~yme-mar~ed T4 is contacted with insoluble and prefer-ably carrier-bound anti-T~ antibody for a time which is sufficiently long to give a constant binding rate of the marked T4 on to the antibody. The period of time depends, to a certain extent, upon the conditions with regard to pH value, temperature and concentration. In general, a period from about 30 minutes to about 2 hours is sufficient. ~en incubation is completed, the sol-ution is separated from the carrier, for example by `
- 9 - ~
pouring out of the container used, the carrier ls washed with water and subsequently the determination of the carrier-bound enzyme activity is carried out. In the case of the preferred embodiment using a container carrier and using POD as the marking enzyme, there is added, for example, hydrogen peroxide and ABTS [2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate)] in a buffer solution, khe extinction difference being deter-mined at a measurement wavelength of 405 nm.
The presenk invention also provide~ a test kit for carrying out the process according to the present invention, which test kit consists essentially of thyroxine, enzyme-marked thyroxine, anti-thyroxine anti-body in the form of insoluble solid material, a bu~fer and a rea~ent for the determination of the enzyme act-ivity. Buffers which can be used are those with which a pH value of from 7.5 to about 9~3 can be adjusted, a pH value of from 8,0 to 9.O being preferredO Typical examples of buffers which can ~e used in the g~ven range include phosph~te buffer, tris buffer, borax buffer and barbital-huffer, 0.05 to 0.5M b~rbital buffer being preferred.
The test kit according to the present invention ~an also contain conventional stabilising agents, such as bovine serum albumin, carbohydrates and/or glycerol, as well as immune reaction-promoking additives.
- As enzyme~marked T4, the test kit according to . J
. .
2~
the present invention preferably contains thyroxine-pero~idase. The reagent for the determination of the enzyme activity can then, in this preferred case, com-prise hydrogen peroxide or a compound which provides hydrogen peroxide, such as sodium perborate or per-hydrite, as well as 2~2'-azino-di-(3-ethylbenzthiazoline-6~sulphonate~, usually referred to as ABTS, and also an appropriate buffer. A typical example of an appropriate buffer is phosphate-citrate buffer (pH 4.5 to 6.0).
Another reagent which can be used for this embodiment comprises phosphate buffer tpH 7.0), guaiacol and hydrogen peroxide.
A special embodiment of the preferred test kit o~
the present invention comprises or contains:
thyroxine 100 - 440 ng./ml.
thyroxine-POD 5 - 100 mU/ml.
barbital buffer (pH 8.6)Ool - 0.2M
bovine serum albumin 0.2%
anti-thyroxine antibody (calculated without carrier) 1 - 0.05 g./ml.
hydrogen peroxide 0.5 - 5 mM/l.
ABTS 5 - 50 mM/l.
phosphate-citrate buffer 0.1 0.2M
For the preparation of the enzyme-marked thyroxine, it is preferable to ~tart from tert.-butylo~ycarbonyl-thyroxine~N-hydroxysuccinimide, the reaction taking place in buffer/dimethylformamide solution (1:1 v/v)~
--ll The buffer used is that which is best suited for the particular enæyme used. Phenylbutylamine-sepharose has proved to be useful for the purification of the conjugate.
Thus, the present invention provides a simple process, which can be carried out with conventional laboratory devices, for the determination of the thyroxine-binding index of serum, the exactitude of which corres-ponds to that of the known methods using radioactive marking but does not suffer from their disadvantages.
The following Example is given for the purpose of illustrating the present invention:-Example.
A. Preparation of anti-thyroxine antibodies.
Thyroxine is coupled to ~ovine serum albumin in aqueous ~olution (pH 10) by the addition of 1.9% by wei~ht glutardialdehyde. The Schiff base bond is then reduced with excess sodium borohydride and the T4 immunogen obtained is purified by chromatography. The product obtained is dialysed and then administered to the experimental animals~
~ .
POD i~ reacted with a 10 fold molar excess of tert.-butyloxycarbonylthyroxine-N-hydroxysuccinimide in dimethylformamide/phosphate buffer lpH 8.5) tl-l v/v).
The dimethylformamide is then dialysed off and the aqueous solution i3 passed over phenylbutyl~mine-sepharose~ The column is washed with tris-sodium chloride buffer and then with 1~ sodium chloride-containiny ethylene glycol/water mi~ture ~1:1 v/v).
The eluate is stirred for 2 hours with 0.5M hydroxylamine and subsequently dialysed against phosphate buffer. The soluti~n obtained i5 mixed with bovine serum albumin and lyophilisedO
C. _reparation of carrier-bound anti-T antibodies.
Anti-T4 antiserum is obtained in known manner from the immunised experimental animals and precipitated with ammonium sulphate. The precipitate is taXen up with 0.04M phosphate buffer 1:6000. 1.5 ml. of the solution so obtained is left to stand overnight in test tubes made of styrerle-acrylonitrile polymer (Luran~, then sucked out and washed with physiological sodium chloride solutio~ which contains 1% BSA and thereafter dried.
nation.
Into the anti-T4 antibody-coated test tubes obtained according to C above are pipetted 10 ~1. serum and sub~equently 1 ml. of a reagent which contains 280 ng T4/ml., 10 mU~ml~ T4-POD in 0.12M barbital buf~er ~pH 8.6) and 0. 2Yo bo~ine serum albumin. After standing ~or 2 hours at ambient ternperature, the test tubes are emptied by sucking out and the POD reagent is placed therein, the latter comprising 1.47 r~M/l.
hydrogen peroxide and 14 mM/l. ABTS in 0~2M phosphate-cit,rate huf~er (pH 5.0). The colour change which occurs is measured at 405 nm.
~ J ~ ~a rl~
For the evaluation, a calibration line is produced with 2 standards of differing T~-~inding capaity which are treated in the same way as the samples, the reciprocal values of the extinctions measured for these standards being plotted either against the T4 amounts bound by the standards or directly against the previously deter-mined TBI values of these standards.
E. Use of the method for the investiqation of human sera.
83 Human sera samples are comparatively investigated y the process according to the~present invention and with method using the use of a commercially available/T3-I125. If the investigated sera are classified on the basis of the particular TBI values obtained with re~erence to the normal range valid for the method in question in those sera with reduced, normal and lncreased T~-binding capacity, then a satisfactory agreement results between both methods (see Table 1).
Comparison of khe process according to the present invention for the determination o~ the TBI (A) with a conventional TBI radio-assay ~B), using human serum samples (n = 83) ~ .
1) reduced 2) normal ) T4-binding capacity
3~ increased) z~
The enzymes referred to in this specification have ~een assigned the following identification nur~ers by the Enzyme Commission:
Peroxidase (1.11.1.7) Glucose-oxidase (1.1.3.4) ~-Galactosidase (-D-Galactosidase) (3.2.1.22) ~-Galactosidase (-D-Galactosidase) (3.2.1.23) 10Glucoarnylase (3.2.1.3) Invertase (3.2.1.26) Alkaline phosphatase (3.1.3.1) C
, ~ ' ' `
.
The enzymes referred to in this specification have ~een assigned the following identification nur~ers by the Enzyme Commission:
Peroxidase (1.11.1.7) Glucose-oxidase (1.1.3.4) ~-Galactosidase (-D-Galactosidase) (3.2.1.22) ~-Galactosidase (-D-Galactosidase) (3.2.1.23) 10Glucoarnylase (3.2.1.3) Invertase (3.2.1.26) Alkaline phosphatase (3.1.3.1) C
, ~ ' ' `
.
Claims (14)
1. A process for the determination of the thyroxine-binding index in serum, wherein a serum sample to be investigated is mixed with a definite amount of thyroxine and with a definite amount of a determinable enzyme covalently bound to thyroxine, the solution obtained is contacted with insoluble anti-thyroxine anti-bodies present in a solid phase, a liquid phase is separated from the solid phase and the activity of the determinable enzyme used is measured in one of the phases.
2. A process according to claim 1, wherein the deter-minable enzyme used is peroxidase.
3. A process according to claim 1, wherein the anti-thyroxine antibody is employed in carrier-bound form.
4. A process according to claim 2, wherein the anti-thyroxine antibody is employed in carrier-bound form.
5. A process according to claim 3 or 4, wherein the carrier is a container having an inner wall coated with the antibody.
6. A process according to claim 1, 2 or 3, wherein 100 to 500 ng. thyroxine/ml. of serum is used.
7. A process according to claim 4, wherein 100 to 500 ng. thyroxine/ml. of serum is used.
8. A process according to claim 2 or 4, wherein 1 to 10mU/ml. of thyroxine-bound peroxidase is used.
9, A process according to claim 4, wherein the solid carrier is a container having an inner wall coated with the antibody, said thyroxine is employed in an amount of 100 to 500 ng. thyroxine/ml. of serum, and said thyroxine-bound peroxidase is employed in an amount of 1 to l0mU/ml.
10. A test kit for the determination of the thyroxine-binding index in serum comprising in combination a container having an inner wall coated with anti-thyroxine antibodies in a solid phase, a reagent com-position for introduction into said container to contact the coated wall, said reagent composition comprising thyroxine, enzyme-marked thyroxine and a buffer; said inner wall being inert to test substances and adapted to support said solid phase and present said solid phase for contact with said reagent composition and a serum sample and permit binding of enzyme-marked thyroxine and thyroxine in said composition to said anti-thyroxine antibodies in said solid phase, and a reagent for introduction to said composition in said container, for the determination of enzyme activity of said solid phase having enzyme-marked thyroxine bound thereto; said container being adapted to hold a sufficient volume of a sample of serum to be determined with a sufficient volume of said reagent composition for said binding, said container being adapted to hold said reagent after expulsion of a reacted mixture of said serum sample and reagent composition for carrying out the determination of said enzyme activity in said container.
11. A test kit according to claim 10, wherein said container is adapted to hold said sample and said reagent composition with a concentration of 100 to 500 ng of thyroxine per ml of serum.
12. A test kit according to claim 10 or 11, wherein said reagent composition further includes at least one of a stabilizing agent and an immune reaction-promoting agent.
13. A test kit according to claim 10, wherein said inner wall is coated with 1 to 0.05 ng/ml of said anti-thyroxine antibodies,and said reagent composition comprises:
thyroxine 100 to 400 ng/ml.
thyroxine-POD 5 to 100 mU/ml.
barbital buffer (pH 8.6) 0.1 to 0.2M
Bovine serum albumin 0.2%, and said reagent comprises:
hydrogen peroxide 0.5 to 5 mM/l.
ABTS 5 to 50 mM/l. and phosphate-citrate buffer (pH 5.0) 0.1 to 0.2M
thyroxine 100 to 400 ng/ml.
thyroxine-POD 5 to 100 mU/ml.
barbital buffer (pH 8.6) 0.1 to 0.2M
Bovine serum albumin 0.2%, and said reagent comprises:
hydrogen peroxide 0.5 to 5 mM/l.
ABTS 5 to 50 mM/l. and phosphate-citrate buffer (pH 5.0) 0.1 to 0.2M
14. A test kit according to claim 10, wherein said inner wall is of polystyrene or a styrene-acrylonitrile copolymer.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP2825650.0 | 1978-06-12 | ||
| DE2825650A DE2825650C3 (en) | 1978-06-12 | 1978-06-12 | Method for determining the thyroxine binding index in serum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1122119A true CA1122119A (en) | 1982-04-20 |
Family
ID=6041575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000326897A Expired CA1122119A (en) | 1978-06-12 | 1979-05-02 | Process for the determination of the thyroxine- binding index in serum |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4467030A (en) |
| EP (1) | EP0006998B1 (en) |
| JP (1) | JPS551590A (en) |
| AT (1) | AT361134B (en) |
| CA (1) | CA1122119A (en) |
| CS (1) | CS208781B2 (en) |
| DE (2) | DE2825650C3 (en) |
| ES (1) | ES481490A1 (en) |
| FI (1) | FI68653C (en) |
| HU (1) | HU182464B (en) |
| YU (1) | YU136479A (en) |
| ZA (1) | ZA792871B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4410633A (en) * | 1980-09-25 | 1983-10-18 | Corning Glass Works | Method for the measurement of free thyroxine or 3,5,3'-triiodothyronine in a liquid sample |
| DE3122019C2 (en) * | 1981-06-03 | 1983-11-10 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method and device for carrying out an automated continuous enzyme immunoassay |
| DE3504406A1 (en) * | 1985-02-08 | 1986-08-14 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETERMINING THE BINDING CAPACITY OF THYROXIN BINDING GLOBULIN |
| DE3546014A1 (en) * | 1985-12-24 | 1987-06-25 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING THE BINDING CAPACITY OF THYROXIN BINDING TBG |
| DE3812610A1 (en) * | 1988-04-15 | 1989-10-26 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING THE THYROXIN BINDING CAPACITY AND SUITABLE STANDARD SOLUTION |
| JP5997446B2 (en) | 2011-02-03 | 2016-09-28 | アークレイ株式会社 | Liquid reagent for immobilizing thyroid hormone and use thereof |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL154598B (en) * | 1970-11-10 | 1977-09-15 | Organon Nv | PROCEDURE FOR DETERMINING AND DETERMINING LOW MOLECULAR COMPOUNDS AND PROTEINS THAT CAN SPECIFICALLY BIND THESE COMPOUNDS AND TEST PACKAGING. |
| US4040907A (en) * | 1974-06-20 | 1977-08-09 | Syva Company | Iodothyronine enzyme conjugates |
| US3962039A (en) * | 1974-08-08 | 1976-06-08 | Center For Laboratory Medicine | Analytical procedure for thyroid hormones |
| US4043872A (en) * | 1975-02-20 | 1977-08-23 | Syva Company | Polyiodothyronine immunoassay |
| US4081245A (en) * | 1976-05-03 | 1978-03-28 | Beckman Instruments, Inc. | Immunoassay procedure employing novel immunochemical composites |
| US4032626A (en) * | 1976-06-30 | 1977-06-28 | Corning Glass Works | Reagent and method for tbg assay |
| US4052504A (en) * | 1976-06-30 | 1977-10-04 | Corning Glass Works | Assay for thyroxine binding globulin |
| GB1573627A (en) * | 1976-09-18 | 1980-08-28 | Ismail A A A | Process and device for the determination and detection of a reaction component |
| US4168207A (en) * | 1977-08-03 | 1979-09-18 | Syva Company | TBG assay |
-
1978
- 1978-06-12 DE DE2825650A patent/DE2825650C3/en not_active Expired
-
1979
- 1979-05-02 CA CA000326897A patent/CA1122119A/en not_active Expired
- 1979-05-21 CS CS793505A patent/CS208781B2/en unknown
- 1979-05-28 AT AT386979A patent/AT361134B/en not_active IP Right Cessation
- 1979-05-29 FI FI791720A patent/FI68653C/en not_active IP Right Cessation
- 1979-06-06 EP EP79101785A patent/EP0006998B1/en not_active Expired
- 1979-06-06 DE DE7979101785T patent/DE2961673D1/en not_active Expired
- 1979-06-11 ZA ZA792871A patent/ZA792871B/en unknown
- 1979-06-11 YU YU01364/79A patent/YU136479A/en unknown
- 1979-06-11 HU HU79BO1790A patent/HU182464B/en not_active IP Right Cessation
- 1979-06-12 JP JP7311379A patent/JPS551590A/en active Granted
- 1979-06-12 ES ES481490A patent/ES481490A1/en not_active Expired
-
1981
- 1981-05-15 US US06/263,948 patent/US4467030A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| HU182464B (en) | 1984-01-30 |
| DE2825650C3 (en) | 1981-02-05 |
| DE2825650B2 (en) | 1980-05-29 |
| JPS551590A (en) | 1980-01-08 |
| ZA792871B (en) | 1980-07-30 |
| US4467030A (en) | 1984-08-21 |
| EP0006998A1 (en) | 1980-01-23 |
| AT361134B (en) | 1981-02-25 |
| FI791720A (en) | 1979-12-13 |
| JPH0146828B2 (en) | 1989-10-11 |
| ATA386979A (en) | 1980-07-15 |
| DE2825650A1 (en) | 1979-12-13 |
| FI68653C (en) | 1985-10-10 |
| FI68653B (en) | 1985-06-28 |
| DE2961673D1 (en) | 1982-02-18 |
| YU136479A (en) | 1984-04-30 |
| ES481490A1 (en) | 1980-01-16 |
| EP0006998B1 (en) | 1981-12-30 |
| CS208781B2 (en) | 1981-09-15 |
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